PROGRAM SUMMARY Mechanical ventilation, a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS), also creates excessive mechanical stress that augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS share many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell (EC) barrier integrity. Insights into VILI pathobiology have been incremental with no viable therapies realized. This PPG intensely focuses on increasing our understanding of: i) the transcription factors that relay the effects of excessive mechanical stress; ii) the molecular signaling pathways that lead to EC injury, including initial activation of a mechanosensitive Ca2+-regulatory receptor, transient receptor potential cation channel subfamily V member 4 (TRPV4); iii) post translational modifications (PTMs) that influence key signaling pathways involved in VILI responses; iv) genetic and epigenetic influences in key target genes involved in VILI responses; and v) novel therapeutic strategies for VILI. The key novel genes that comprise the focus of each Project were identified by our genomic?intensive approaches and selected for their capacity to contribute to a spectrum of VILI responses from VILI-induced lung inflammation, increased vascular permeability and injury (Projects #1 and #2); to VILI resolution with restoration of lung vascular barrier integrity (Project #3). These strategies are integrated across our three PPG projects and represent the thematic underpinnings of this PPG. Studies will be conducted by an outstanding group of gifted and interactive translational scientists. Project #1 will examine the NF-?B- dependent mechanisms (including protein nitration) by which VILI downregulates expression of SOX18, a critical lung vascular barrier-protective transcription factor (TF), and the key tight junction protein, claudin 5. The influence of the mechanosensitive receptor, TRPV4 on mitochondrial ROS and mechanical stress-associated TFs such as HIF2? will be explored. Project #2 will extend novel insights regarding the critical role of secreted extracellular NAMPT (eNAMPT), a nicotinamide phosphoribosyltransferase, in VILI and ARDS. Excessive mechanical stress induces NAMPT expression and eNAMPT ligates TLR4 (Toll-like receptor 4) to induce NF-?B signaling and inflammatory lung injury. Project #2 will interrogate novel mechanisms of NAMPT secretion, the influence of NAMPT/TLR4 SNPs, and NAMPT and TLR4 as therapeutic targets. Project #3 will interrogate genetic and epigenetic regulation of mechanical stress-mediated sphingosine 1-phosphate receptor expression (S1PR1, S1PR3) and the role of VILI-induced nitration of Rac1 and RhoA GTPases in lung vascular barrier regulation. The synergy derived from the interaction between individual Projects, as well as with our scientific Cores, with enviable expertise in molecular biology (B), genetic epidemiology (B), pre-clinical models of disease (C), and protein chemistry & Biophyics (D), will advance our programmatic approaches and promote the development of novel, individualized therapies to attenuate VILI especially in populations at risk for ARDS.